ABSTRACT
Follicle-stimulating hormone (FSH) activates FSH receptors (FSHR) in granulosa cells to induce follicle differentiation, growth and estradiol production. FSH is used clinically to treat female infertility and is administered by injection. To increase patient convenience and compliance, compound homogeneity and composition, low molecular weight (LMW), orally bioavailable, FSHR agonists are now being developed to replace FSH. In this study, we present the signaling mechanisms of a newly developed LMW dihydropyridine agonist of the FSHR, Org 214444-0. Org 214444-0 is shown to be a stereoselective, nanomolar potent FSHR agonist and selective over the structurally related LHR and TSHR. Org 214444-0 is an allosteric agonist interacting with the transmembrane region of the FSHR. When co-incubated with FSH, Org 214444-0 augments FSH's potency in binding (6.5-fold) and adenylyl cyclase/cAMP activation (3.5-fold) in a concentration-dependent manner. Like FSH, Org 214444-0 induces FSHR internalization and is only marginally effective in stimulating phospholipase C. Moreover, Org 214444-0 stimulates cAMP and estradiol production in human granulosa cells in culture and supports the follicular phase in mature female rats. We conclude that Org 214444-0 is a bonafide FSHR agonist.
Subject(s)
Dihydropyridines/pharmacology , Receptors, FSH/agonists , Sulfonamides/pharmacology , Allosteric Regulation , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , Cyclic AMP/physiology , Female , Follicle Stimulating Hormone/metabolism , Molecular Sequence Data , Molecular Weight , Rats , Receptors, FSH/chemistry , Signal Transduction , Type C Phospholipases/metabolismABSTRACT
BACKGROUND AND PURPOSE: Graves' disease (GD) is an autoimmune disease in which the thyroid is overactive, producing excessive amounts of thyroid hormones, caused by thyroid-stimulating hormone (TSH) receptor-stimulating immunoglobulins (TSIs). Many GD patients also suffer from thyroid eye disease (Graves' ophthalmopathy or GO), as TSIs also activate TSH receptors in orbital tissue. We recently developed low molecular weight (LMW) TSH receptor antagonists as a novel therapeutic strategy for the treatment of GD and GO. Here, we determined the molecular pharmacology of a prototypic, nanomolar potent LMW TSH receptor antagonist, Org 274179-0. EXPERIMENTAL APPROACH: Using CHO cells heterogeneously expressing human TSH receptors and rat FRTL-5 cells endogenously expressing rat TSH receptors, we determined the potency and efficacy of Org 274179-0 at antagonizing TSH- and TSI-induced TSH receptor signalling and its cross-reactivity at related follicle-stimulating hormone and luteinizing hormone receptors. We analysed the allosteric mode of interaction of Org 274179-0 and determined whether it is an inverse agonist at five naturally occurring, constitutively active TSH receptor mutants. KEY RESULTS: Nanomolar concentrations of Org 274179-0 completely inhibited TSH (and TSI)-mediated TSH receptor activation with little effect on the potency of TSH, in accordance with an allosteric mechanism of action. Conversely, increasing levels of TSH receptor stimulation only marginally reduced the antagonist potency of Org 274179-0. Org 274179-0 fully blocked the increased basal activity of all the constitutively active TSH receptor mutants tested with nanomolar potencies. CONCLUSIONS AND IMPLICATIONS: Nanomolar potent TSH receptor antagonists like Org 274179-0 have therapeutic potential for the treatment of GD and GO.
